Factor IX plays a pivotal role in both the extrinsic and intrinsic pathways of bolld coagulation. Establishment of the regualtory mechanisms underlying factor IX (FIX) biosynthesis, therefore, is critical for understanding not only the molecular basis of hemophilia B, but also the role of FIX in the blood coagulation and its homeostasis. The long-term goal of this proposal is to establish the comprehensive control mechanism responsible for FIX gene transcription, the first step in the FIX biosynthesis. This will be accomplished by achieving three specific aims, (I) to establish the precise fundamental mechanism responsible for the overall transcriptional control of the FIX gene; (ii) to establish specific mechanisms responsible for the puberty-onset amelioration of the hemophilia B Leyden phenotype; and (iii) to establish the molecular basis underlying the age-associated regulation of FIX gene expression. Aim 1 will achieved by establishing a detailed map of essential structural elements, their functions and mutual relationship n the overall regulation of the FIX gene, thus providing a solid base for the rest of the proposed studies. Systematic in vitro identification and functional analysis of cis-acting DNA sequence elements and trans-acting factors interacting with them will be carried out by site-directed mutagenesis, nuclease hypersensitivity assay, DNA- protein and protein-protein interaction analyses as well as characterization of crucial trans-acting factors, if they are novel. In vivo, functions of these elements in context of FIX minigene structures will tested in transgenic mice designed in conjunction with the studies for aims 2 and 3. Aim 2, mechanisms responsible for the hemophilia B Leyden phenotype, which are due to a set of single-base mutations contained in a small 5' end region, will be studied by extending our new findings including possible involvement of growth hormone pathway, with intensive in vitro as well as in vivo analyses including testing with transgenic mice combined with hypophysectomy. We have recently found a novel role of the Line 1 sequence present at the 5' end in the age-associated FIX gene regulation. Aim 3 will achieved by establishing the mechanism of action of the sequence in context of the rest of gene structure. In this study, transgenic mice will serve as an important in vivo assay system. Proposed studies will significantly contribute to our understanding of the FIX biosynthesis in specific, and the overall regulation of blood coagulation and its homeostasis in general, and this to the development of improved or perhaps novel teratments for bleeding and thrombotic disorders in humans.